Method and apparatus for making density gradients

ABSTRACT

A float is used for preparing a density gradient in a parallel-walled vessel. The float has an outer peripheral surface that has a diameter smaller than an inner diameter of an inner surface of the vessel. With the float placed inside the vessel a liquid is introduced onto the float such that the liquid flows around the float between the float and the inner wall of the vessel. The shape and configuration of the float slows the velocity of the liquid such that there is only laminar flow as the liquid contacts other liquid below the float. Elimination of turbulent flow prevents mixing of different liquid introduced into the same vessel thereby forming layers of fluid. Preferably, the vessel is a centrifuge tube. In one embodiment, the outer diameter of the float is large enough to cause capillary action between the float and the inner surface of the centrifuge tube to force liquid to remain between the float and the inner surface of the centrifuge tube.

BACKGROUND OF THE INVENTION

[0001] A. Field of the Invention

[0002] The present invention relates to an apparatus and method formaking a multiple density layers or gradients of fluid in a vessel in ahighly reproducible manner using a float that floats on the surface ofthe fluid within the vessel.

[0003] B. Description of the Related Art

[0004] There are various fields where it is desirable to have densitylayers or gradients of fluid within a vessel for such purposes as theseparation of matter, determining density, etc. Such density layersinclude, for example, a solution retained in a vessel where the fluid isdivided into a plurality of layers, each layer having differingconcentrations of a soluble material or solute. For example, a bottom orfirst layer of fluid may have a concentration of a solute that is Xmoles per liter; a second layer immediately above the first layer mayhave a concentration of 0.8 X moles per liter; a third layer above thesecond layer may have a concentration of 0.6 X moles per liter; and afourth layer having a concentration of 0.4 X moles per liter.

[0005] Liquids having gradients of temperature, concentration, densityand color have been previously prepared. Liquid density gradients havebeen used for many years, for a wide variety of purposes, in a number ofdifferent industries. The inventor has numerous publications and patentsregarding certain aspects of gradient formation and use includingAnderson, N. G. Mechanical device for producing density gradients inliquids. Rev. Sci. Instr. 26: 891-892, 1955; Anderson, N. G., Bond, H.E., and Canning, R. E. Analytical techniques for cell fractions. I.Simplified gradient elution programming. Anal. Biochem. 3: 472-478,1962; Anderson, N. G., and Rutenberg, E. Analytical techniques for cellfractions. A simple gradient-forming apparatus. Anal. Biochem. 21:259-265, 1967; Candler, E. L., Nunley, C. E., and Anderson, N. G.Analytical techniques for cell fractions. VI. Multiplegradient-distributing rotor (B-XXI). Anal. Biochem. 21: 253-258, 1967.

[0006] A variety of other methods for making density gradients have beendeveloped, and Bock, R. M. and Ling, N.-S., Anal. Chem. 26, 1543, 1954,and Morris, C. J. O. R, and Morris, P., Separation Methods inBiochemistry, Pitman Publishing, 2nd ed. (1976) have reviewed many ofthese. Only one of these methods allowed gradients to be made frommultiple solutions, each having a different combination of reagents(Anderson, et al, “Analytical Techniques for Cell Fractions. I.Simplified Gradient Elution Programming”, Analytical Biochemistry 3:472-478, 1962) More recent innovations include the use of pumps andpistons, which are differentially controlled by microprocessors, e.g.,the Angelique gradient maker (Large Scale Proteomics Corp. Rockville,Md.). Gradients may also be generated during high speed centrifugationby sedimenting a gradient solute such as cesium chloride or an iodinatedx-ray contrast medium such as iodixanol. Gradients may be initiallyprepared as step gradients and linearized by diffusion, by gentlemixing, or by freezing and thawing. A list of references coveringexisting methods follows.

[0007] Density gradients are used to make two basic types ofseparations. The first separates particles on the basis of sedimentationrate (rate-zonal centrifugation), in which case particles are separatedon the basis of the size and density (and to a lesser extent shape) andparticles will sediment farther if centrifuged for a longer period oftime. The second separates particles on the basis of isopycnic bandingdensity, in which case particles reach their equilibrium density level,and do not sediment farther with continued centrifugation.

[0008] Four types of gradients are in general use with either of thesebasic methods. The first includes step gradients, made by layering aseries of solutions of decreasing density (if the solutions areintroduced one above the other), and of increasing density (if thesolutions are introduced sequentially to the bottom of the tube). Thesecond type comprises linear continuous gradients usually made by amechanical gradient maker. These are usually introduced slowly throughsmall tubing to the bottom of the centrifuge tube. Linear gradients foreither rate zonal or isopycnic zonal centrifugation are useful forresolving very heterogeneous mixtures of particles.

[0009] The third type of gradient is non-linear, and may be designed toseparate particles having a very wide range of sizes or densities.Non-linear gradient may be designed to separate particles on the basisof both sedimentation rate and isopycnic banding density in the samegradient, in which case some particles reach their isopycnic level atsome point in the gradient, while others are still sedimenting.Generally such combined separations involve larger and denser particleswhich band near the bottom of the gradient, while other smaller, andusually lighter particles are still sedimenting in the upper portion ofthe gradient.

[0010] The fourth type of gradient is generated in a high centrifugalfield by sedimentation of the major gradient solute, and is usually usedfor isopycnic banding.

[0011] Many reasons exist for desiring to control gradient shape.Gradient capacity (i.e., the mass of particles which can exist in a zonewithout causing a density inversion) is a function of gradient slope,and a steep gradient can support a greater mass of particles per unitgradient length than can shallow gradients. The greatest particle massconcentration in a gradient separation usually occurs immediatelybeneath the sample zone shortly after centrifugation is started. Asdifferent particles separate in the length of the gradient, thepossibility of an overloaded zone diminishes. For this reason it isdesirable to have a short steep gradient section immediately under thesample zone, where the highest gradient capacity is required.

[0012] An additional reason for desiring to control gradient shape isthat when a population of particles is present that differ little insedimentation rate, these can best be separated by sedimentation througha longer shallower section of the gradient. Such shallow sections areusually near the center of a gradient.

[0013] In the majority of density gradient separations, the gradientsand their chemical composition are designed to optimize the separationof one or a few particles types. This accounts for the very large numberof different gradient recipes that have been published for subcellularfractionation. Those used for the isolation of mitochondria, forexample, are usually quite different from those used to isolate nuclei.For example, traces of divalent cations are required to control nuclearswelling, whereas such ions are generally deleterious to othersubcellular particles. Low concentrations of nonionic detergents removecytoplasmic contamination from nuclei, but are deleterious to theendoplasmic reticulum. Hence there has been no one procedure or gradientthat has been optimized for the systematic separation of the majority ofall subcellular particles. There is a need for reproducible means forincluding in gradients zones containing salts, detergents, enzymes andother reactive substances that would increase the number of differentsubcellular particles separated in one gradient.

[0014] Density gradient separations are important in proteomicsresearch. High resolution two-dimensional electrophoresis (2DE) iswidely used to produce global maps of the proteins in extracts preparedby solubilizing whole cells or tissues. By careful control of theprocedures employed, use of staining procedures which are quantitative,and computerized image analysis and data reduction, quantitativedifferences in the abundance of individual proteins of ±15% has beenachieved (Anderson, N. Leigh, Nance, Sharron L., Tollaksen, Sandra L.,Giere, Frederic A., and Anderson, Norman G., Quantitativereproducibility of measurements from Coomassie Blue-stainedtwo-dimensional gels: Analysis of mouse liver protein patterns and acomparison of BALB/c and C57 strains. Electrophoresis 6: 592-599, 1985;Anderson, N. Leigh, Hofmann, Jean-Paul, Gemmell, Anne, and Taylor, John,Global approaches to quantitative analysis of gene-expression patternsobserved by use of two-dimensional gel electrophoresis. Clin. Chem. 30:2031-2036, 1984). There is a need for precision subcellularfractionation that will allow changes in abundance of minor proteins tobe accurately detected and measured in data which sums the abundance ofall proteins found in all of the fractions of one sample.

[0015] This technology allows changes in gene expression, as reflectedin protein abundance, to be studied under a wide range of conditions,and has led to the development of databases of protein abundance changesin response to a wide variety of drugs, toxic agents, disease states. Insuch studies large sets of data must be acquired and intercompared.Hence all stages in one pharmaceutical study, for example, must bestandardized for intercomparability.

[0016] 2DE maps of whole cells or tissues typically contain a thousandor more protein spots in sufficient abundance to allow each protein tobe analyzed by mass spectrometry and identified and characterized.However, it is known that a very much larger number of proteins areactually present in tissue samples analyzed than are actually observed.The number present varies with cell or tissue type, and is believed tobe up to ten or twenty times the number detected.

[0017] Different subcellular particles and the soluble fraction of thecell (the cytosol) contain many location-specific proteins whichconstitute only trace fractions of the total cell protein mass. Hencethe total number of proteins resolved from one cell type or tissue couldbe greatly increased if the 2DE analysis were done on cell fractionsrather than on whole cell or tissue extracts as has previously beendemonstrated (Anderson, N. L., Giere, F. A., et al, Affects of toxicagents at the protein level: Quantitative measurements of 213 mouseliver proteins following xenobiotic treatment. Fundamental and Appl.Tox. 8 :39-50, 1987). If a drug effect study is to be done on cellfractions, however, the fractionation procedures must be quantitative,in the sense that the same organelles, or even mixtures of organellesare used in all analyses to be intercompared. There exists, therefore,an emerging need for high resolution density gradient separations usingprecision gradients in proteomics research. Making precision gradientsreproducibly and in parallel has proven to be difficult, particularlywhen the gradients are shallow.

[0018] The protein composition of tissues such as liver variesdiurnally, hence all the tissues from one group of animals are preparedat the same time of day, and, to be comparable, must be fractionated inparallel, on the same time schedule, and, if gradients are to be used,in identical gradients. Further, gradient fraction recovery must also bedone from all gradients in parallel, under identical conditions. If theinitial separations are done partly or entirely on a sedimentation ratebasis, and if the recovered fractions are to then each be isopycnicallybanded, as is done in two-dimensional or s-ρ fractionation, then thesesubsequent steps must also be carried out in parallel. This, in turn,requires that the gradients be made in parallel.

[0019] Precision gradients are difficult to make in practice, and it isfurther difficult to confirm that a set of gradients are all identicalwithout destroying them for analysis. Existing swinging bucket rotorsgenerally allow six gradients to be centrifuged simultaneously. Largernumbers may be centrifuged if the lower resolution of vertical or nearvertical tube rotors is accepted. Therefore if existing density gradientformers are to be used, a set of six or more of them operating inparallel will be required.

[0020] With any gradient maker, small amounts of turbulence ornon-laminar flow typically cause solutions of differing concentrationsto at least partially mix, thereby reducing the effectiveness andusefulness of the density layers. There is therefore a need for a methodfor decelerating fluids flowing into a tube, and for moving them slowlyinto position to form distinct bands.

[0021] One of many uses of density layers and gradients is in the fieldsof cell separation, sub-cellular fractionation and analysis, and densitygradient methods are used in molecular biology and in polymer chemistry.Little attention has been paid to forming sets of precision-madegradients that are highly reproducible for cell separation. There istherefore a requirement for precision gradients adapted to cellseparation.

[0022] One high resolution system is disclosed in “Development of ZonalCentrifuges”, by N. G. Anderson, National Cancer Inst. Monograph 21,1966) and employs zonal centrifuge rotors. The rotors are of highcapacity, and process one sample at a time. However, the rotor volumesare too high for many applications. Angle head or vertical rotor tubesmay also be employed (Sheeler, P., Centrifugation in Biology andMedicine, Wiley Interscience, N.Y., 1981, 269pp) using either step orcontinuous gradients. However these do not provide the resolutionobtained with swinging bucket rotors.

[0023] There has been no reliable method for reproducibly locating andrecovering organelle zones purely on the basis of the physicalparameters of sedimentation rate and isopycnic banding density.Mathematical analyses, based on analysis not only of the biologicalparticles separated, but of the gradients themselves have been required.These have been tedious and idiosyncratic to the rotors and conditionsemployed. The basic problem in preparing density gradients in tubes isthat the liquid volume elements of either step (layers), or continuousgradients must be introduced into tubes very slowly or mixing willoccur. This problem is only partially overcome by introducing thegradient into a set of tubes in an angle-head rotor during rotation.

[0024] Methods for producing one or a few gradients in parallel havebeen developed, but fraction recovery is generally done one at a time.The gradients are rarely identical, and it is difficult to introduce thesample layer on top of the gradient without mixing. Hence there is nopublished data on the quantitative high-resolution protein analysis ofcell fractions of animals subject to various experimental treatments. Ifmultiple, parallel identical gradients are to be prepared using gradientengines (for instance, see “Mechanical device for producing densitygradients in liquids” by N. G. Anderson,. Rev. Sci. Instruments 26:891-892, 1955) one must have one machine for each tube being filled.Centrifugal gradient distributing heads have been built (see “A MethodFor Rapid Fractionation of Particulate Systems by Gradient DifferentialCentrifugation” by J. F. Albright, and N. G. Anderson, Exptl. CellResearch 15: 271-281, 1958), however the gradients actually producedtend to be uneven, and a refrigerated centrifuge is required. There is,therefore, a continuing need for simple gradient makers that produceidentical gradients in parallel in sufficient number to satisfy currentrequirements. There is a further need for a simple, disposable andeasily sterilizable system for making reproducibly sharp step gradients.An additional need exists for a system or device that can produce verynarrow-step density gradients in which diffusion can rapidly andreproducibly even out the steps. A further need exists for a system ordevice which allows individual gradient steps to be rapidly pipettedinto centrifuge tubes, either manually or robotically, and in which theintroduced fluid does not disturb the underlying gradient. A stillfurther need exists for a gradient making device in which thecomposition of the successive layers, while forming a stable densityseries, differ in composition relative to salts, enzymes, detergents orother reactive materials.

SUMMARY OF THE INVENTION

[0025] One object of the present invention is to provide a rapid, simpleand reproducible method and apparatus for forming a multiplicity ofliquid density gradients in vessels.

[0026] Another object of the present invention is to provide a rapid,simple and reproducible method and apparatus for forming a multiplicityof liquid density gradients in vessels for rate-zonal separations, forisopycnic banding separations, or a combination of the two.

[0027] Yet another object of the present invention is to provide anapparatus and method for reproducibly producing a plurality of liquiddensity gradients in a plurality of corresponding vessels, each vesselhaving a specific predetermined liquid density gradient.

[0028] An additional object of the present invention is to provide meansfor making liquid density gradients in which aliquots of a liquiddensity series are rapidly pipetted into the centrifuge tubes withoutregard to potential stirring or mixing.

[0029] A further object of the invention is to decelerate the aliquotsejected from pipettes or automatic pipetters, and to cause them to flowevenly into position without disturbing the underlying fluids.

[0030] A further object of the present invention is to provide means formaking the linear or complex gradients by making them initially as stepgradients having very small density differences per step.

[0031] A further object of the present invention is to produce stepgradients in which the steps are so small that diffusion rapidly evensout the gradient.

[0032] A still further object of the present invention is to make thegradient making components disposable and easily sterilizable.

[0033] It is a further object of the present invention to make possibleconstruction of sets of identical gradients in a short period of time.

[0034] It is an additional object of the present invention to makepossible addition of the sample layer on top of the gradient at any timeafter the gradient is formed.

[0035] In accordance with one aspect of the present invention, there isa method for producing liquid density gradients in a vessel using afloat within the vessel includes the steps of:

[0036] inserting the float in the vessel;

[0037] introducing a first liquid into the vessel;

[0038] introducing a second liquid into the vessel such that the secondliquid contacts at least one surface of the float upon entering thevessel, contact between surfaces of the float and the second liquidallowing the second liquid to form a layer above the first liquidthereby forming separate layers of liquid; and

[0039] repeating the second introducing step with successive introducingsteps with a third, fourth and so on liquid.

[0040] The float used in the above method slows the velocity of fluidsuch that flow of liquid is laminar thereby limiting mixing of the twoliquids.

[0041] In accordance with another aspect of the present invention, anapparatus for producing liquid density gradients includes a vessel and afloat positionable in the vessel. The float is formed with at least onesurface that is shaped to inhibit acceleration of fluid introduced intothe vessel thereby restricting turbulent flow of the fluid.

[0042] An outer peripheral surface of the float and the inner surface ofthe vessel are sized such that in response to fluid being introducedinto the vessel above the float, the fluid undergoes capillary actionmoving downward beneath the float in the vessel.

BRIEF DESCRIPTION OF THE DRAWINGS

[0043] Further advantages of the disclosed invention will becomeapparent from a reading of the following description when read inconjunction with the accompanying drawings where like reference numeralsare used to identify like parts, in which:

[0044]FIGS. 1A, 1B, 1C, 1D, 1E, 1F, and 1G are side views of a vesseland a float for producing a density gradient in the vessel in accordancewith the present invention;

[0045]FIGS. 2A, 2B and 2C show details of the design and operation offloat;

[0046]FIGS. 3A, 3B and 3C are side views showing alternate embodimentsof the float;

[0047]FIGS. 4A and 4B are a side view showing yet another embodiment ofthe float; and

[0048]FIG. 5 is a side view showing still another embodiment of thefloat.

DETAILED DESCRIPTION OF THE INVENTION

[0049] A first embodiment of the present invention is illustrated inFIGS. 1A, 1B, 1C, 1D, 1E, 1F, and 1G. In accordance with the presentinvention, a float 1 is used to form a step gradient within a vessel 2,as depicted in FIGS. 1A, 1B, 1C, 1D, 1E, 1F, and 1G. However, it shouldbe understood that the float 1 may also be used to create a continuousgradient (not shown) where a density gradient is introduced gradually,and continuously changes along the height of the vessel. In FIG. 1A thefloat 1 in the vessel 2 rises and floats on top of a liquid 3 introducedfrom a source 4. The diameter of the float 1 in the first embodiment ispreferably slightly smaller than the inside diameter of the vessel 2.

[0050] The introduced liquid 3 contacts and then flows around the float1, passes down between the outer surface of the float 1 and the innersurface of the vessel 2 and, as shown in FIG. 1A, produces a first zone5. Typically, the zone 5 has the highest density of all the layers orzones, as is described in greater detail below. As shown in FIG. 1B, asecond liquid 6 is introduced in a similar manner to produce zone 7. Asshown in FIGS. 1C, 1D, and 1E, the procedure is repeated with succeedingless dense liquids 8, 10, and 12, to produce zones 9, 11, and 13.

[0051] A sample 14 to be analyzed within the vessel 2 is introduced lastfrom a pipette 15 to produce zone 16 as shown in FIGS. 1F and 1G.Finally as shown in FIG. 1G, the float 1 is removed by grasping aprojecting pin 17 and lifting. The vessel 2 depicted in FIG. 1G withsample and gradient may then be subjected to treatment by, for instance,insertion into a swinging bucket rotor of a centrifuge device. When acontinuous gradient is required, the step gradient is preparedbeforehand, and diffusion for a determined period of time used toconvert the step gradient into a continuous one.

[0052]FIGS. 2A, 2B and 2C illustrate details of the float 1 and basicprinciples of operation of the float within the vessel 2. The float 1and vessel 2 depicted in FIG. 2A is shown enlarged in FIG. 2B. The float1 has an outer peripheral surface 20 that is shaped to conform to aninner surface of the vessel 2. For instance, the vessel 2 shown in thedrawings is a tube having a circular cross-section when viewed from topor bottom. The float 1 has a corresponding circular shape with the outerperipheral surface 20 having a diameter that is smaller than the innerdiameter of the surface of the vessel 2. Therefore, a gap G having apredetermined width is defined between the outer peripheral surface 20of the float 1 and the inner surface of the vessel 2. The gap G may varyin size depending upon the solutions to be introduced into the vessel 2and the relative sizes of the float 1 and vessel 2.

[0053] In the depicted embodiment, the gap G is relatively small suchthat surface tension of the solution produces a capillary action withinthe gap G to maintain liquid in the gap and to prevent air bubbles inthe gap. In many applications of the present invention the viscosity offluid in the gap is sufficient to eliminate the possibility of turbulentfluid flow within the vessel 2 as the solution exits the gap and movesaround the float 1. Therefore, mixing of layers of solution under thegap is almost non-existent and very sharp boundaries are producedbetween the zones, even with very small density increments.

[0054] It should be understood that diminished rate of fluid flow is adesirable result of the present invention depicted in FIGS. 1A-1G andFIGS. 2A-2C. The actual size of the gap G may be varied according to theviscosity of the liquids used and the size of the vessel 2. However,although capillary action and restricted rate of fluid flow areimportant to the present invention, it is possible to use the float 1 ofthe present invention without capillary action. For instance, the shapeof the surfaces of the float 1 may be formed to discourage any increasesin velocity of fluid moving over the surfaces of the float 1 to avoidturbulent flow of the fluids entering the vessel 2. The shape andsurface contours of the float 1 are such that the flow of solutionaround the float 1 as the solution moves downward into the vessel 2 isminimal. Specifically, a upper surface 22 of the float 1 is taperedhaving a conical shape such that as fluid contacts the upper surface 22viscous flow slows fluid motion as the fluid approaches an edge 23 ofthe float 1.

[0055] It should be understood that the upper surface 22 may have a morerounded shape when viewed from the side and need not be conical in shapeso long as sufficient surface area is provided to allow the adhesiveforces of the fluid to make contact with the upper surface 22 to slowmovement of the fluid.

[0056] It should also be understood that the vessel 2 and float 1 mayhave any of a variety of shapes when viewed in cross-section. Thedepicted vessel 2 is a tube having a circular cross-section. The vessel2 may also have a square or triangular cross-sectional shape and thefloat 1 a corresponding square or triangular cross-sectional shape.

[0057] As shown in FIG. 2B, when a droplet 21 of solution is droppedfrom above the float 1, the droplet 21 is distributed circumferentiallyon upper tapered surface 22 and moves toward the edge 23, where thesolution flows evenly into the gap G, and thereafter slowly moves on tothe upper surface of the underlying layer of liquid. Velocity or speedof flow of the solution is also further decelerated as it flows aroundlower taper 25. Capillary forces and solution viscosity are sufficientto keep the velocity of the solution in the gap G to a minimum andfurther, regardless of the density of the liquids used, the gap Gtypically remains filled with solution due to the capillary action.

[0058] The float 1 is also formed with an upper integral pin 17 thatallows the float 1 to be inserted and removed easily from the vessel 2.The density of the float 1 itself may be dictated by the choice ofconstruction material or, as shown in FIG. 2C, an alternate embodimentof a float 1 a may be formed with a cavity 26 sealed by plug 27 toadjustably control the density of the float 1. The floats 1 and 1 a arepreferentially constructed of polypropylene which has a density ofapproximately 0.95 g/cc, and the pin 17, being a small fraction of themass of the float, may be either integrally molded into the float and ofthe same material, or may be another material such as polycarbonate orother plastic, and be inserted in a hole in the float as shown in FIGS.4A and 4B. Further, the density of the float may be adjusted byinserting pins 17 having a variety of weights. For instance, a pluralityof pins 17 may be produced, each pin 17 having a different mass forselectively adjusting the overall weight of the float.

[0059] The shape of the various surfaces of the float 1 is not limitedto the depictions in FIGS. 1A-1G and FIGS. 2A-2C. FIGS. 3A, 3B and 3Cillustrate alternative float designs. In FIG. 3A a float 28 has upperedges 29 and lower edges 30 rounded to further assist in slowlyaccelerating flow at the upper edge, and decelerating flow at the loweredge as liquid flows over the underlying liquid. In FIG. 3B float 31 hasdifferent upper edge 32 and lower edge 33, with the upper edge 32 sharpto help prevent air bubbles between the float and tube 2, and the loweredge 33 well rounded, while in FIG. 3C float 34 has a tip 35 extended tofurther control flow around the float. The shape of the lower surface ofthe float 34 and the tip 35 assist in keeping the interface 36 betweentwo steps in the gradient sharp.

[0060] The inventors have tested and designed floats for BeckmanUltraclear tubes for the Beckman SW41 Ti rotor and for polycarbonatetubes for the Beckman SW28 rotor. For the SW 41 tubes, the floats wereconstructed of solid polypropylene, 13.1 mm in diameter with top andbottom tapers of 15 degrees, and were 6.35 mm high measured at the edge.Wall clearance was 0.25 mm (gap G). For the SW 28 rotor tubes, long andshort versions of the floats were constructed which were 10.5 mm and 6mm high at the edge, had clearances of 1 and 0.6 mm, with 15 degreetapers at the top and bottom. Holes through the float had 1.6 mminternal diameters, and the pins were made of 0.9 mm outside diameterpolycarbonate monofilament. After the pins were inserted, one end wasmelted in a reducing flame to produce a ball at the tip, while the otherend was heated to produce a small enlargement, which, when put into thefloat, sealed the pin in place.

[0061] All radial clearances kept the gap G between the float and thecentrifuge tube wall (vessel 2) full of liquid at all densities used.Occasionally when floats were dropped into dry tubes, they became stuckat the bottom, hence the “round” at the bottom of the centrifuge tube ispreferably filled with a “cushion”, i.e., densest gradient solutionused, initially.

[0062] Experimentally it was found that if the first 4-5 drops (circa0.1 ml) of the solution being added were introduced slowly over a periodof 5-10 seconds, extraordinarily sharp interfaces were produced belowthe float. The remainder of the gradient step could then be introducedmore rapidly. Sharp interfaces were produced with the density differencebetween two steps being as little as 0.0017 g/ml.

[0063] The use of floats allows gradients to be formed as a series ofshort well defined zones that may be arranged to be linear, sigmoidal,or of other gradient shape. If required, the gradients can then beevened by diffusion. The float/vessel arrangement allows the productionof gradients that are more reproducible than those produced byconventional gradient makers, and allows many gradients to be made inparallel without requiring a multiplicity of gradient makers.

[0064] However, it should also be understood that by using the float ofthe present invention, it is possible to quickly pour an amount of afluid directly onto the top of the float and the fluid will graduallyseep down around the float to create a layer fluid without significantlydisturbing the layer or layers of fluid already beneath the float.Without the float, pouring of fluid into the vessel with previouslyintroduced fluid layers in the vessel would guarantee mixing of thelayers thereby making gradient layer formation impossible. Therefore,one important result possible by using any of the above describedembodiments of the present invention is that a density gradient can beproduced quickly and reproducibly without concern of the rate of flow ofany one liquid onto the upper surface of the float.

[0065] In yet another embodiment of the present invention depicted inFIG. 5, a float 50 is positioned in a vessel 52 with the vessel 52having an inner diameter that is significantly greater than the outerdiameter of the float 50. The float 50 is similar to the float describedabove in FIGS. 1A-1G, but has a tube 55 attached to an upper surface ofthe float 50. The tube 55 is hollow and includes several apertures 58for allowing the flow of fluid from within the tube 55 to an uppersurface of the float 50. As fluid is introduced from the tube 55 via theapertures 58, the fluid contacts the upper surface of the float 50 andflows along the upper surface due to adhesion thereby slowly enteringthe vessel. Adhesion between the surfaces of the float 50 and the fluidslows velocity of the fluid such that the fluid forms a well definedlayer above previously introduced layers.

[0066] The upper surface 51 of the float 50 is preferably formed withonly a slight incline to further inhibit acceleration of the fluid. Thetube 55 attached to the fluid may also be used to raise and lower thefloat 50 with respect to the vessel 52. Specifically, a mechanical armmay be attached to the tube 55 to remotely control movement of the floatin and out of the vessel 51. It should be understood that the tube 55 isflexible to allow movement of the float 50 upward as the vessel 52 isfilled with fluid.

[0067] A fluid flow controller (not shown) is preferably used with theembodiment of the float 50 to control the amount of fluid introduced foreach desired layer.

[0068] It should be understood that gradients may be used in manyapplications outside of the field of molecular biology. For instance, avessel having a plurality of layers solution, each layer having adifferent density due to a specific concentration of solute in eachlayer, may be used determine the density of an unknown material. Asample of material of unknown density dropped into the vessel willsettle in the layer having a like density thereby providing a means fordetermining density of the unknown material. For example, differentclasses of plastics have different densities. Small pieces of plasticmay easily be tested by dropping one small sample into a vessel having aplurality of solutions, each solution having a predetermined densitysuch that the plurality of layers define a stepped density gradient. Theplastic particle will drop to a layer having the same density and willfloat above those layers having a heavier density. Similarly,identification of a gemstone based on density can be conducted.

[0069] The materials and methods of the instant invention can be used inthe separation of cellular elements from samples of whole blood, bloodproducts or diluted blood. For example, white blood cells can beobtained from blood by density gradient centrifugation. Suitablematerials to effect separation of the cellular elements and particularlythe nucleated cellular elements from blood include media that comprisecolloidal silica, silica gel, sugars, such as sucrose, ficoll, andparticular products such as Ficoll-Hypaque, Isopaque, LymphoPrep andPercoll. See, for example, Parish et al., Eur. J. Imm. (1974) 4:808.

[0070] Generally single step gradients are produced by gently layeringthe blood cell suspension onto a high density medium. The preparationthen is centrifuged at low speed to effect separation of the cells.

[0071] Alternatively, the blood cell suspension can be layered onto alinear gradient, for example, of bovine serum albumin prior tocentrifugation. The blood cell suspension can be layered onto adiscontinuous gradient, for example, of bovine serum albumin. Thedensities of the layers can be configured so that the various elementsband at the interfaces of the layers.

[0072] To ensure that discrete, sharp layers and hence tight banding ofcells occurs, it is beneficial to ensure a sharp interface between thecell suspension, for example, blood, and the separation medium. Thatgoal can be achieved with use of an apparatus of interest. A suitablysized float of interest is used. The float of interest rests atop theseparation medium. The float of interest allows passage of the, forexample, blood along the lateral sides thereof and along the innersurface of the centrifuge tube containing the medium, float and cellsuspension with minimal turbulence to ensure formation of a discretelinear interface of cell suspension and separation medium.

REFERENCES

[0073] Anderson, N. G., ed. The Development of Zonal Centrifuges.National Cancer Institute Monograph 21, 1966, 256 pp,.

[0074] Price, C. A. Centrifugation in Density Gradients. Academic Press,New York 1982 430 pp.

[0075] Scheeler, P. Centrifugation in Biology and Medical Science. JohnWiley & Sons New York 1981 269pp.

[0076] Anderson, N. G. A simple method for observing refractive indexgradients in liquids. Biochim. Biophys. Acta 25: 418, 1957.

[0077] Albright, J. F., and Anderson, N. G. A method for the rapidfractionation of particulate systems by gradient differentialcentrifugation. Exptl. Cell Research 15: 271-1181, 1958.

[0078] Anderson, N. G., Bond, H. E., and Canning, R. E. Analyticaltechniques for cell fractions. I. Simplified gradient elutionprogramming. Anal. Biochem. 3: 472-478, 1962.

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[0135] All references cited herein are herein incorporated by referencein entirety.

[0136] Although the present invention has been described with referenceto the preferred embodiments, the invention is not limited to thedetails thereof. Various substitutions and modifications will occur tothose of ordinary skill in the art and all such substitutions andmodifications are intended to fall within the scope of the invention asdefined in the appended claims.

What is claimed is:
 1. A method for producing liquid density gradientsin a vessel using a float within the vessel, the method comprising thesteps of: inserting the float in the vessel; introducing a first liquidinto the vessel; and introducing a second liquid into the vessel suchthat the second liquid contacts at least one surface of the float uponentering the vessel, contact between surfaces of the float and thesecond liquid allowing the second liquid to form a layer above the firstliquid thereby forming separate layers of liquid.
 2. The method as setforth in claim 1 further comprising: introducing a third liquid into thevessel such that the third liquid contacts at least one surface of thefloat upon entering the vessel, contact between surfaces of the floatand the third liquid allowing the third liquid to form a third layerabove the second liquid thereby forming three separate layers of liquid.3. The method as set forth in claim 2 further comprising: introducing afourth liquid into the vessel such that the fourth liquid contacts atleast one surface of the float upon entering the vessel, contact betweensurfaces of the float and the fourth liquid allowing the fourth liquidto form a fourth layer above the second liquid thereby forming fourseparate layers of liquid.
 4. The method as set forth in claim 2 whereinthe first liquid has a density greater than the density of the secondand third liquids and the density of the second liquid is greater thanthe third liquid.
 5. The method as set forth in claim 1 wherein in saidinsertion step, the float is surrounded by an inner surface of thevessel such that during subsequent steps, the various liquids undergocapillary action contacting both an outer peripheral surface of thefloat and the inner surface of the vessel as the fluid is drawn bygravity under the float.
 6. An apparatus for producing liquid densitygradients, the apparatus comprising: a vessel; and a float positionablein said vessel, said float being formed with at least one surface thatis shaped to inhibit acceleration of fluid introduced into said vesselthereby restricting turbulent flow of the fluid.
 7. The apparatus as setforth in claim 6 , wherein an outer peripheral surface of said float hasa shape conforming to an inner surface of said vessel.
 8. The apparatusas set forth in claim 7 wherein said vessel and said outer peripheralsurface of said float have a round shape.
 9. The apparatus as set forthin claim 7 wherein said vessel and said outer peripheral surface of saidfloat have a generally square shape.
 10. The apparatus as set forth inclaim 7 wherein said vessel and said outer peripheral surface of saidfloat have a triangular shape.
 11. The apparatus as set forth in claim 7wherein said outer peripheral surface of said float and said innersurface of said vessel are sized such that in response to fluid beingintroduced into said vessel above said float, the fluid undergoescapillary action moving downward beneath said float in said vessel. 12.The apparatus as set forth in claim 6 , wherein said vessel is acentrifuge tube.
 13. A float for use in preparing a density gradient ina parallel-walled vessel wherein said float has an outer peripheralsurface having a diameter smaller than an inner diameter of an innersurface of said vessel, and, with said float placed into said vessel andin response to liquid being placed onto said float the liquid flowsaround said float between said float and said inner wall of said vessel.14. A float as set forth in claim 13 , wherein said vessel is acentrifuge tube.
 15. A float as set forth in claim 14 wherein said outerdiameter of said float is large enough to cause capillary action betweensaid float and said inner surface of said centrifuge tube to forceliquid to remain between said float and said inner surface of saidcentrifuge tube.
 16. A float as set forth in claim 14 wherein said floathas a conical upper surface which tapers down from a central apex to anupper edge of said outer peripheral surface.
 17. A float as set forth inclaim 16 wherein said outer peripheral surface has a lower edge and saidfloat has a lower surface that tapers downward from said lower edge to acentral portion thereof.
 18. A float as set forth in claim 17 whereinsaid upper edge and said lower edge are rounded over.
 19. A float as setforth in claim 17 wherein said upper edge has a sharp edge.
 20. A floatas set forth in claim 17 wherein said central portion of said lowersurface includes a pointed region.
 21. A float as set forth in claim 16further comprising a pin fixed to said float at said central apex.
 22. Afloat as set forth in claim 13 wherein said float comprises a plasticmaterial having a density less than water such that said float floats ona water based solution.
 23. A float as set forth in claim 13 whereinsaid float comprises polypropylene.
 24. A float as set forth in claim 13wherein said float comprises polyethylene.
 25. A float as set forth inclaim 13 wherein said float is formed with a central cavity having avolume sufficient to reduce the buoyant density of said float to lessthan that of water.
 26. A method for preparing a gradient in a vessel,comprises the steps of: inserting the float of claim 13 into the vessel;introducing a liquid into the vessel above the float such that theliquid flows around the float into a portion of the vessel under thefloat.
 27. A method as set forth in claim 26 , wherein the float isinserted into the vessel prior to addition of any fluids to the vessel.28. A method as set forth in claim 26 , comprising the step of:introducing a first liquid into the vessel before inserting the floatinto the vessel, the first liquid forming a first layer of the gradientand the liquid introduce in the second step in claim 26 forming a secondlayer of the gradient.
 29. A method as set forth in claim 26 , whereinthe gradient a continuous gradient.
 30. A method as set forth in claim26 , wherein the gradient comprises a plurality of layers of liquid,each liquid having decreasing densities.
 31. A method as set forth inclaim 29 , further comprising the steps of: introducing a second liquidinto the vessel above the float such that the second liquid flows aroundthe float into a portion of the vessel under the float and above thepreviously introduced liquid; introducing a third liquid into the vesselabove the float such that the third liquid flows around the float into aportion of the vessel under the float and above the previouslyintroduced second liquid; and allowing the vessel to sit a predeterminedtime interval sufficient to allow partial diffusion of the layers ofliquid to convert a step gradient into a semi-continuous gradient. 32.An apparatus for isolating nucleated cells from blood comprising: (a) avessel containing a blood cell separating medium; and (b) a floatpositionable in said vessel, said float being formed with at least onesurface that is shaped to inhibit acceleration of a blood sampleintroduced into said vessel, thereby restricting turbulent low of saidblood sample onto said separating medium.
 33. The apparatus of claim 32, wherein said medium comprises silica.
 34. The apparatus of claim 32 ,wherein said medium comprises a sugar.
 35. The apparatus of claim 32 ,wherein said medium comprises ficoll.
 36. An apparatus for isolatingnucleated cells from a blood sample comprising: (a) a vessel containinga blood cell separating medium; and (b) the float of any one of claims13-25, wherein said liquid is a blood sample.
 37. A method for isolatingnucleated cells from blood comprising: (a) introducing a blood sampleinto the apparatus of claim 32 ; and (b) centrifuging said sample insaid vessel to produce a gradient, wherein nucleated cells separate andform a discrete layer in said gradient.